This patent application claims a benefit of priority from Korean Patent Application No. 2000-44972 filed Aug. 3, 2000 and Korean Patent Application No. 2001-46911 filed Aug. 3, 2001, the contents of each of which are incorporated herein by reference.
The present invention relates to an inhibitor against replicative senescence of human keratinocytes. The inhibitor against replicative senescence of human keratinocytes can extend the in vitro life span and inhibit replicative senescence. Therefore, the inhibitor containing retinoic acid as active ingredients of the present invention can be used for a prophylactic or therapeutic agent of various oral diseases, such as trauma-caused inflammation, exelcymosis-caused inflammation, burn-caused inflammation, traumatic ulcer, and angular cheilosis, for a cosmetic purpose and for a prophylactic or therapeutic agent for wound-caused dermatitis and skin senescence.
Replicative senescence is regarded to be an essential causative element of organismic aging (Wantanabe Y. et al. Oncogene, 1997, 14, 2025-2032). It is assumed that such senescent cells are accumulated in the living body and the phenotype expressed from the cells alters thereby accumulating in age-related pathology (Dimri G. D., et al., Proc. Natl. Acad. Sci., 1995, 92, 9363-9367).
It is shown that replicative senescence is dependent upon creative cell division, which indicates that a xe2x80x9cmitotic clockxe2x80x9d limits cell proliferation (Bodnor A. G. et al., Science, 998, 279, 349-352). It was reported that random damage caused by synthesis of RNA or protein, accumulated mutations or alternatively a genetically programmed process resulted in replicative senescence (Sugawara O. et al., Science, 1990, 247, 707-710).
Additionally, cell cycle regulatory proteins and tumor suppressors are known to have influence on replicative senescence. Actually, it has been certified that increased cellular levels of wild-type p53 and p21WAP1/Cip1 proteins induce to the loss of call proliferative capacity in some cultured cells, which indicates cellular senescence (Brown J. P. et al., Science, 1997, 277, 831-834; Gallimore P. H et al., Cell Growth Differ. 1997, 8, 763-771; Sugrue M. Mm. et al., Proc. Natl. Acad. Sci., 1997, 94, 9648-9653).
p16INK4A protein, a G1 cyclin-dependent kinase (Cdk) suppressor, has also been associated with cellular senescence in a variety of cell types (Alcorta D. A. et al Proc. Natl. Acad. Sci., 1996, 93, 13742-13747; Loughran O. et al., Oncogene, 1996, 13, 561-568; Reznikoff C. A. et al, Cancer Res., 1996, 56, 2886-2890; Palmero I. et al. Oncogene, 1997, 15, 495-503; Uhrbom L. et al., Oncogene 1997, 15, 505-514). In particular, p16INK4A was found to form a complex with both cdk4 and cdk6 in senescent cells. Based on this result, it is believed that the regulatory phenomena, which increases intracellular p16INK4A protein levels, is recognized as the most important molecular change that is observed at the growth-stopped final stage of the aging process where all the growth stops. Also, the p16INK4A protein levels in senescent human fibroblasts are elevated compared to that of young fibroblasts, and the p16INK4A protein is often mutated or deleted in immortal cell lines (Vojta P. et al., Biochem. Biophys. Acta., 1995, 1242, 29-41).
pRb, a tumor suppressor gene product, has also indicated the senescent state in human fibroblasts. The p16INK4A protein can be negatively controlled by pRb at the transcription stage. Also, it is found that neither cyclin D/cdk4 nor cyclin D/cak6 is formed in pRb-deficient cells, and thus all Cdk associate wish excessively expressed p16INK4A in senescent cells. This indicates that pRb is related to senescence.
Moreover, the activation of phosphatidylinositol-specific phospholipase C may also be related to cellular senescence in particular cell types (Choudhury G. G. et al., FEBS Lett., 1991; 293, 211-214).
Meanwhile, the animal epithelial cells are known as have a limited proliferative capacity hang M. K. al., Cell Growth Differ., 1998, 9, 85-95; Min B. -M. et al., Exp. Cell Res 1999, 249, 377-385; Dimri G. D. et al., Proc. Natl. Acad. Sci., 1995, 92, 9363-9367), and thus enter the step of replicative senescence after a finite number of cell divisions.
The characteristic features of replicative senescence can be observed through culturing keratinocctes. The senescent cells stop growing, and do not enter the S phase of the ell cycle, regardless of any similar stimulation of mitogen, and inhibit levels of some genes required for cell cycling. However, the senescent cells maintain metabolic activity as usual and the ability to synthesize protein or RNA for a predetermined period of time (Dimri C-. D. et al., Proc. Natl. Acad. Sci., 1995. 92, 9363-9367; Saunders N. A. et al, Biochem. Biophy. Res. Commun., 1993, 197, 46-54).
Recently it is discovered that young normal human keratinocytes maintain their telomerase activity but the senescent cells lose their activity outstandingly It can be inferred from this fact that there is a positive relation between telomerase activity and replicative senescence of cells (Harle-Bachor C. et al, Proc. Natl. Acad. Sci. 1996, 93. 6476-6481).
The above-mentioned human epithelial cells are classified into human mucosal keratinocytes and human epidermal keratinocytes The human epithelial cells have the similar function as a protective epithelium responsible for functionally covering the skin surface. However, physically, there is a great difference. This is human epidermal keratinocytes include well-developed thick cornified cell layers, whereas the human mucosal keratinocytes have relatively thin cornified cell layers, or do not have any cornified cell layers at all (e.g., oral sulcular epithelium and junctional epithelium). In addition to differences of the physical form, human mucosal keratinocytes and human epidermal keratinocytes show a considerable difference in chemical properties and protein expression levels.
Retinoic acid, an analogue of vitamin A, plays a fundamental role in embryogenesis, reproduction, vision, and tie control of cell growth, besides other normal differentiation of many adult tissues (Sauret J. H. et al., Horm. Res., 1995, 43, 89-92).
A topical administration of retinoic acid to a portion of human skin improves several aspects of photoaged skin, including fine and deep wrinkles texture, and color and it has also been found to inhibit the induction of a number of metalloproteinases involved in the destruction of the extracellular matrix (Varani J. et al., Am. J. Pathol. 1990, 136, 1275-1281; Varani J. et al., J. Invest. Dermatol. Symp. Proc., 1998, 3, 57-60). In addition, retinoic acid also reduces the differentiation of keratinocyte, induces the deposition of glycosaminoglycan-like material in the epidermis, increases cellularity in the dermis, effects the synthesis new extracellular matrix and fibroblast activation (Varani J. et al., Skin Pharmacol., 1991, 4, 254-261).
However, the effect of retinoic acid, which controls the replicative senescence of human epithelial cells on proliferation, replicative senescence, and the expression of genes, have not been reported yet.
The present invention leads to giving an intensive and thorough research of retinoic acid, which result in the finding that retinoic acid affected expression of genes and activity of proteins controlling cell proliferation and/or replicative senescence; that is a life span of both the human mucosal keratinocytes and the human epidermal keratinocytes can be extended.
Therefore, the object of the present invention is to provide an inhibitor against replicative senescence of human keratinocytes containing retinoic acid as active ingredients.
The human keratinocytes according to present invention include human mucosal keratinocytes, preferably including the human oral keratinocytes(NHOK), and human epidermal keratinocytes (NHEK).
Another object of the present invention is to provide a prophylactic or therapeutic agent of various oral diseases, such as trauma-caused inflammation, exelymosis-caused inflammation, barn-caused inflammation, traumatic ulcer, and angular cheilosis, which contains retinoic acid as active ingredients.
The object of the present invention is to provide a cosmetic purpose by containing retinoic acid as active ingredients.
It is a further object of the present invention to provide a prophylactic or therapeutic agent for wound-caused dermatitis and skin senescence, which contains retinoic acid as active ingredients.